Interleukin-1 receptor intracellular ligand proteins

ABSTRACT

Novel IL-1-R intracellular ligand proteins are disclosed. Polynucleotides encoding the IL-1-R intracellular ligand protein are also disclosed, along with vectors, host cells, and methods of making the IL-1-R intracellular ligand protein. Pharmaceutical compositions containing the IL-1-R intracellular ligand protein, methods of treating inflammatory conditions, and methods of inhibiting IL-1-R intracellular domain binding are also disclosed. Methods of identifying inhibitors of IL-1-R intracellular domain binding and inhibitors identified by such methods are also disclosed.

This application is a divisional of application Ser. No. 08/487,942,filed Jun. 7, 1995.

BACKGROUND OF THE INVENTION

The present invention relates to the field of anti-inflammatorysubstances and other substances which act by inhibiting binding to theintracellular domain of an interleukin-1 receptor (hereinafter"IL-1-R"), such as, for example, the p80, type I IL-1 receptor. Moreparticularly, the present invention is directed to novel ligands whichbind to the IL-1-R intracellular domain and to inhibition or modulationof signal transduction by this receptor.

Interleukin-1-α and interleukin-1-β (herein collectively "IL-1") arecytokines which produce a wide range of cellular activities. IL-1 causesan inflammatory response, which can be beneficial, such as in mountingan immune response to a pathogen, or when overexpressed can lead toother detrimental effects of inflammation.

The cellular effects of IL-1 are initiated by the binding of IL-1 to itsreceptors (IL-1-Rs) on the surface of target cells. The isolation ofpolynucleotides encoding IL-1-Rs and variant forms of such receptors hasbeen described in U.S. Pat. Nos. 4,968,607, 5,081,228, 5,180,812, in PCTPublication No. WO91/18982, and by Sims et al., PNAS, 86, 8946 (1989)(disclosing the p80, type I IL-1 receptor). Processes for purificationof IL-1-Rs have also been disclosed in U.S. Pat. No. 5,296,592.

Native IL-1-Rs are characterized by distinct extracellular,transmembrane and intracellular domains. The primary purpose of theextracellular domain is to present a binding site for IL-1 on theoutside of the cell. When IL-1 is bound to the binding site, a "signal"is transmitted to the inside of the cell through the transmembrane andintracellular domains, indicating that binding has occurred.Transmission or "transduction" of the signal to the inside of the celloccurs by a change in conformation of the transmembrane and/orintracellular domains of the receptor. This signal is "received" by thebinding of proteins and other molecules to the intracellular domain ofthe receptor, resulting in the effects seen upon IL-1 stimulation.

While IL-1 binding by IL-1-Rs results in beneficial cellular effects, itis often desirable to prevent or deter IL-1 binding from causing otherdetrimental cellular effects. Although substantial effort has beenexpended investigating inhibition of IL-1 binding to the extracellulardomain of IL-1-Rs, examination of binding of proteins and othermolecules to the intracellular domain of IL-1-Rs has received much lessattention.

However, ligands which bind to the IL-1-R intracellular domain have yetto be identified. It would be desirable to identify and isolate suchligands to examine their effects upon IL-1-R signal transduction andtheir use as therapeutic agents for treatment of IL-1-inducedconditions. Furthermore, identification of such ligands would provide ameans for screening for inhibitors of IL-1-R/intracellular ligandbinding, which will also be useful as anti-inflammatory agents.

SUMMARY OF THE INVENTION

Applicants have for the first time identified novel IL-1-R intracellularligand proteins and have isolated polynucleotides encoding such ligands.Applicants have also identified certain known proteins which may alsobind to the intracellular domain of IL-1-R.

In one embodiment, the present invention provides a compositioncomprising an isolated polynucleotide encoding a protein having IL-1-Rintracellular ligand protein activity. In preferred embodiments, thepolynucleotide is selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1from nucleotide 2 to nucleotide 529;

(b) a polynucleotide comprising a fragment of the nucleotide sequence ofSEQ ID NO:1, which encodes a protein having IL-1-R intracellular ligandprotein activity;

(c) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:2;

(d) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising a fragment of the amino acid sequence of SEQ ID NO:2 andhaving IL-1-R intracellular ligand protein activity;

(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3from nucleotide 2 to nucleotide 961;

(f) a polynucleotide comprising a fragment of the nucleotide sequence ofSEQ ID NO:3, which encodes a protein having IL-1-R intracellular ligandprotein activity;

(g) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:4;

(h) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising a fragment of the amino acid sequence of SEQ ID NO:4 andhaving IL-1-R intracellular ligand protein activity;

(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5from nucleotide 2 to nucleotide 754;

(j) a polynucleotide comprising a fragment of the nucleotide sequence ofSEQ ID NO:5, which encodes a protein having IL-1-R intracellular ligandprotein activity;

(k) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:6;

(l) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising a fragment of the amino acid sequence of SEQ ID NO:6 andhaving IL-1-R intracellular ligand protein activity; and

(m) a polynucleotide capable of hybridizing under stringent conditionsto any one of the polynucleotides specified in (a)-(l), which encodes aprotein having IL-1-R intracellular ligand protein activity.

In certain preferred embodiments, the polynucleotide is operably linkedto an expression control sequence. The invention also provides a hostcell, including bacterial, yeast, insect and mammalian cells,transformed with such polynucleotide compositions.

Processes are also provided for producing an IL-1-R intracellular ligandprotein, which comprises:

(a) growing a culture of the host cell transformed with suchpolynucleotide compositions in a suitable culture medium; and

(b) purifying the IL-1-R intracellular ligand protein from the culture.

The ligand protein produced according to such methods is also providedby the present invention.

Compositions comprising a protein having IL-1-R intracellular ligandprotein activity are also disclosed. In preferred embodiments theprotein comprises an amino acid sequence selected from the groupconsisting of:

(a) the amino acid sequence of SEQ ID NO:2;

(b) fragments of the amino acid sequence of SEQ ID NO:2;

(c) the amino acid sequence of SEQ ID NO:4;

(d) fragments of the amino acid sequence of SEQ ID NO:4;

(e) the amino acid sequence of SEQ ID NO:6; and

(f) fragments of the amino acid sequence of SEQ ID NO:6;

the protein being substantially free from other mammalian proteins. Suchcompositions may further comprise a pharmaceutically acceptable carrier.

Compositions comprising an antibody which specifically reacts with suchIL-1-R intracellular ligand protein are also provided by the presentinvention.

Methods are also provided for identifying an inhibitor of IL-1-Rintracellular domain binding which comprise:

(a) combining an IL-1-R intracellular domain protein with an IL-1-Rintracellular ligand protein, said combination forming a first bindingmixture;

(b) measuring the amount of binding between the IL-1-R intracellulardomain protein and the IL-1-R intracellular ligand protein in the firstbinding mixture;

(c) combining a compound with the IL-1-R intracellular domain proteinand an IL-1-R intracellular ligand protein to form a second bindingmixture;

(d) measuring the amount of binding in the second binding mixture; and

(e) comparing the amount of binding in the first binding mixture withthe amount of binding in the second binding mixture;

wherein the compound is capable of inhibiting IL-1-R intracellulardomain binding when a decrease in the amount of binding of the secondbinding mixture occurs. In certain preferred embodiments the IL-1-Rintracellular ligand protein used in such method comprises an amino acidsequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:2;

(b) fragments of the amino acid sequence of SEQ ID NO:2;

(c) the amino acid sequence of SEQ ID NO:4;

(d) fragments of the amino acid sequence of SEQ ID NO:4;

(e) the amino acid sequence of SEQ ID NO:6;

(f) fragments of the amino acid sequence of SEQ ID NO:6;

(g) the amino acid sequence of SEQ ID NO:7; and

(h) fragments of the amino acid sequence of SEQ ID NO:7.

Compositions comprising inhibitors identified according to such methodare also provided. Such compositions may include pharmaceuticallyacceptable carriers.

Methods are also provided for preventing or ameliorating an inflammatorycondition which comprises administering a therapeutically effectiveamount of a composition comprising a protein having IL-1-R intracellularligand protein activity and a pharmaceutically acceptable carrier.

Other embodiments provide methods of inhibiting IL-1-R intracellulardomain binding comprising administering a therapeutically effectiveamount of a composition comprising a protein having IL-1-R intracellularligand protein activity and a pharmaceutically acceptable carrier.

Methods of preventing or ameliorating an inflammatory condition or ofinhibiting IL-1-R intracellular domain binding are provided, whichcomprise administering to a mammalian subject a therapeuticallyeffective amount of inhibitors of IL-1-R intracellular domain binding,are also provided.

Methods of identifying an inhibitor of IL-1-R intracellular domainbinding are also provided by the present invention which comprise:

(a) transforming a cell with a first polynucleotide encoding an IL-1-Rintracellular domain protein, a second polynucleotide encoding an IL-1-Rintracellular ligand protein, and at least one reporter gene, whereinthe expression of the reporter gene is regulated by the binding of theIL-1-R intracellular ligand protein encoded by the second polynucleotideto the IL-1-R intracellular domain protein encoded by the firstpolynucleotide;

(b) growing the cell in the presence of and in the absence of acompound; and

(c) comparing the degree of expression of the reporter gene in thepresence of and in the absence of the compound;

wherein the compound is capable of inhibiting IL-1-R intracellulardomain binding when a decrease in the degree of expression of thereporter gene occurs. In preferred embodiments, the cell is a yeast celland the second polynucleotide is selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1from nucleotide 2 to nucleotide 529;

(b) a polynucleotide comprising a fragment of the nucleotide sequence ofSEQ ID NO:1, which encodes a protein having IL-1-R intracellular ligandprotein activity;

(c) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:2;

(d) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising a fragment of the amino acid sequence of SEQ ID NO:2 andhaving IL-1-R intracellular ligand protein activity;

(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3from nucleotide 2 to nucleotide 961;

(f) a polynucleotide comprising a fragment of the nucleotide sequence ofSEQ ID NO:3, which encodes a protein having IL-1-R intracellular ligandprotein activity;

(g) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:4;

(h) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising a fragment of the amino acid sequence of SEQ ID NO:4 andhaving IL-1-R intracellular ligand protein activity;

(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5from nucleotide 2 to nucleotide 754;

(j) a polynucleotide comprising a fragment of the nucleotide sequence ofSEQ ID NO:5, which encodes a protein having IL-1-R intracellular ligandprotein activity;

(k) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:6;

(l) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising a fragment of the amino acid sequence of SEQ ID NO:6 andhaving IL-1-R intracellular ligand protein activity;

(m) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:7;

(n) a polynucleotide encoding an IL-1-R intracellular ligand proteincomprising a fragment of the amino acid sequence of SEQ ID NO:7 andhaving IL-1-R intracellular ligand protein activity; and

(o) a polynucleotide capable of hybridizing under stringent conditionsto any one of the polynucleotides specified in (a)-(n), which encodes aprotein having IL-1-R intracellular ligand protein activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an autoradiograph demonstrating the expression of IL-1-Rintracellular ligand proteins of the present invention in mammaliancells. The expression of flag-14w, -31w and -19w was detected by ananti-flag antibody, M2, as described below.

FIG. 2 demonstrates the effects of the clone 19w product on JNK1activation. Top panel: HA-tagged JNK1 was coexpressed in COS cells witheither pED flag vector of pED flag-19w. After 48 hr, the cells weretreated with different concentrations of IL-1α for 15 min. JNK1 wasisolated by immunoprecipitation with 12CA5 antibody and JNK activity wasmeasured using an immune complex kinase assay with the substrateGST-c-jun (1-79). Middle panel: The expression and recovery if HA-JNK1from immunoprecipitation was examined by Western blot analysis wit 12CA5antibody. Bottom panel: The expression of clone 19w was detected byWestern blot analysis of cell lysate using M2 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have for the first time identified and isolatednovel polynucleotides which encode proteins which bind to the IL-1-Rintracellular domain. As used herein "IL-1-R" includes all receptors forinterleukin-1. The type I, p80 IL-1-R is the preferred receptor forpracticing the present invention.

The sequence of a polynucleotide encoding one such protein is set forthin SEQ ID NO:1 from nucleotide 2 to 529. This polynucleotide has beenidentified as "clone 19w." The amino acid sequence of the IL-1-Rintracellular ligand protein encoded by clone 19w is set forth in SEQ IDNO:2. It is believed that clone 19w is a partial cDNA clone of a longerfull length coding sequence. However, as demonstrated herein the proteinencoded by clone 19w does bind the intracellular domain of IL-1-R (i.e.,has "IL-1-R intracellular ligand protein activity" as defined herein).Clone 19w was deposited with the American Type Culture Collection onMar. 31, 1995 and given the accession number ATCC 69774. The proteinencoded by clone 19w is 176 amino acids in length. No identical orclosely related sequences were found using database searches. Therefore,clone 19w encodes a novel protein. However, using an extensive FASTAsearch, a significant homology to amino acids 330 to 390 ofthrombospondin (41% identity in 59 amino acids) is found in theC-terminal portion of the 19w protein. Moreover, a significant homologyto the Ca²⁺ binding domain, EF hand of calmodulin (25% in 65 aminoacids) is observed in the region between amino acids 40 and 110 of theprotein encoded by clone 19w.

The sequence of a polynucleotide encoding another such protein is setforth in SEQ ID NO:3 from nucleotide 2 to 961. This polynucleotide hasbeen identified as "clone 31w." The amino acid sequence of the IL-1-Rintracellular ligand protein encoded by clone 31w is set forth in SEQ IDNO:4. It is believed that clone 31w is a partial cDNA clone of a longerfull length coding sequence. However, as demonstrated herein the proteinencoded by clone 31w does bind the intracellular domain of IL-1-R (i.e.,has "IL-1-R intracellular ligand protein activity" as defined herein).Clone 31w was deposited with the American Type Culture Collection onMar. 31, 1995 and given the accession number ATCC 69775. The proteinencoded by clone 31 w is 320 amino acids in length. No identical orclosely related sequences were found using BLASTN/BLASTX or FASTAsearches. Therefore, clone 31w encodes a novel protein.

The sequence of a polynucleotide encoding another such protein is setforth in SEQ ID NO:5 from nucleotides 2 to 754. This polynucleotide hasbeen identified as "clone 14w." The amino acid sequence of the IL-1-Rintracellular ligand protein encoded by clone 14w is set forth in SEQ IDNO:6. It is believed that clone 14w is a partial cDNA clone of a longerfull length coding sequence. However, as demonstrated herein the proteinencoded by clone 14w does bind the intracellular domain of IL-1-R (i.e.,has "IL-1-R intracellular ligand protein activity" as defined herein).CLone 14w was deposited with the American Type Culture Collection onMar. 31, 1995 and given the accession number ATCC 69773.

The protein encoded by clone 14w is identical to the sequence of aminoacids 449 to 700 of calcium activated neutral protease (CANP), with theexception of an amino acid change (Val to Phe) at position 553 of CANP.The sequence of CANP is disclosed in Imajoh et al., Biochemistry 1988,27, 8122-8128, which is incorporated herein by reference (accession no.A31218). The amino acid sequence of CANP is set forth in SEQ ID NO:7.Based upon this sequence homology, CANP and certain fragments thereofwill exhibit IL-1-R intracellular ligand binding activity (as definedherein).

For the purposes of the present application, "IL-1-R intracellularligand protein" includes proteins which exhibit IL-1-R intracellularligand protein activity. For the purposes of the present application, aprotein is defined as having "IL-1-R intracellular ligand proteinactivity" when it binds to a protein derived from the IL-1-Rintracellular domain. Activity can be measured by using any assay whichwill detect binding to an IL-1-R intracellular domain protein. Examplesof such assays include without limitation the interaction trap assaysand assays in which IL-1-R intracellular domain protein which is affixedto a surface in a manner conducive to observing binding, includingwithout limitation those described in Examples 1 and 3. As used hereinan "IL-1-R intracellular domain protein" includes the entireintracellular domain or fragments thereof.

Fragments of the IL-1-R intracellular ligand protein which are capableof interacting with the IL-1-R intracellular domain or which are capableof inhibiting IL-1-R intracellular domain binding (i.e., exhibit IL-1-Rintracellular ligand protein activity) are also encompassed by thepresent invention. Fragments of the IL-1-R intracellular ligand proteinmay be in linear form or they may be cyclized using known methods, forexample, as described in H. U. Saragovi, et al; Bio/technology 10,773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114,9245-9253 (1992), both of which are incorporated herein by reference.Such fragments may be fused to carrier molecules such as immunoglobulinsfor many purposes, including increasing the valency of IL-1-Rintracellular ligand protein binding sites. For example, fragments ofthe IL-1-R intracellular ligand protein may be fused through "linker"sequences to the Fc portion of an immunoglobulin. For a bivalent form ofthe IL-1-R intracellular ligand protein, such a fusion could be to theFc portion of an IgG molecule. Other immunoglobulin isotypes may also beused to generate such fusions. For example, an IL-1-R intracellularligand protein--IgM fusion would generate a decavalent form of theIL-1-R intracellular ligand protein of the invention.

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the IL-1-R intracellular ligand proteinrecombinantly. Many suitable expression control sequences are known inthe art. General methods of expressing recombinant proteins are alsoknown and are exemplified in R. Kaufman, Methods in Enzymology 185,537-566 (1990). As defined herein "operably linked" means that theisolated polynucleotide of the invention and the expression controlsequence are situated within a vector or cell in such a way that theIL-1-R intracellular ligand protein is expressed by a host cell whichhas been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the IL-1-R intracellular ligand protein. Host cells include, forexample, monkey COS cells, Chinese Hamster Ovary (CHO) cells, humankidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3cells, CV-1 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK orJurkat cells.

The IL-1-R intracellular ligand protein may also be produced by operablylinking the isolated polynucleotide of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

Alternatively, it may be possible to produce the IL-1-R intracellularligand protein in lower eukaryotes such as yeast or in prokaryotes suchas bacteria. Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the IL-1-R intracellularligand protein is made in yeast or bacteria, it may be necessary tomodify the protein produced therein, for example by phosphorylation orglycosylation of the appropriate sites, in order to obtain thefunctional IL-1-R intracellular ligand protein. Such covalentattachments may be accomplished using known chemical or enzymaticmethods.

The IL-1-R intracellular ligand protein of the invention may also beexpressed as a product of transgenic animals, e.g., as a component ofthe milk of transgenic cows, goats, pigs, or sheep which arecharacterized by somatic or germ cells containing a nucleotide sequenceencoding the IL-1-R intracellular ligand protein.

The IL-1-R intracellular ligand protein of the invention may be preparedby culturing transformed host cells under culture conditions suitable toexpress the recombinant protein. The resulting expressed protein maythen be purified from such culture (i.e., from culture medium or cellextracts) using known purification processes, such as gel filtration andion exchange chromatography. The purification of the IL-1-Rintracellular ligand protein may also include an affinity columncontaining the IL-1-R intracellular domain or other IL-1-R intracellulardomain protein; one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GASepharose®; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography.

Alternatively, the IL-1-R intracellular ligand protein of the inventionmay also be expressed in a form which will facilitate purification. Forexample, it may be expressed as a fusion protein, such as those ofmaltose binding protein (MBP) or glutathione-S-transferase (GST). Kitsfor expression and purification of such fusion proteins are commerciallyavailable from New England BioLab (Beverly, MA) and Pharmacia(Piscataway, N.J.), respectively. The IL-1-R ligand protein can also betagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope ("Flag") iscommercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the IL-1-R intracellular ligand protein. Someor all of the foregoing purification steps, in various combinations, canalso be employed to provide a substantially homogeneous isolatedrecombinant protein. The IL-1-R intracellular ligand protein thuspurified is substantially free of other mammalian proteins and isdefined in accordance with the present invention as an "isolated IL-1-Rintracellular ligand protein."

IL-1-R intracellular ligand proteins may also be produced by knownconventional chemical synthesis. Methods for constructing the proteinsof the present invention by synthetic means are known to those skilledin the art. The synthetically-constructed protein sequences, by virtueof sharing primary, secondary or tertiary structural and/orconformational characteristics with IL-1-R intracellular ligand proteinsmay possess biological properties in common therewith, including IL-1-Rintracellular ligand protein activity. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedIL-1-R intracellular ligand proteins in screening of therapeuticcompounds and in immunological processes for the development ofantibodies.

The IL-1-R intracellular ligand proteins provided herein also includeproteins characterized by amino acid sequences similar to those ofpurified IL-1-R intracellular ligand proteins but into whichmodification are naturally provided or deliberately engineered. Forexample, modifications in the peptide or DNA sequences can be made bythose skilled in the art using known techniques. Modifications ofinterest in the IL-1-R intracellular ligand protein sequences mayinclude the replacement, insertion or deletion of a selected amino acidresidue in the coding sequence. For example, one or more of the cysteineresidues may be deleted or replaced with another amino acid to alter theconformation of the molecule. Mutagenic techniques for such replacement,insertion or deletion are well known to those skilled in the art (see,e.g., U.S. Pat. No. 4,518,584).

Other fragments and derivatives of the sequences of IL-1-R intracellularligand proteins which would be expected to retain IL-1-R intracellularligand protein activity in whole or in part and may thus be useful forscreening or other immunological methodologies may also be easily madeby those skilled in the art given the disclosures herein. Suchmodifications are believed to be encompassed by the present invention.

IL-1-R intracellular ligand protein of the invention may also be used toscreen for agents which are capable of inhibiting or blocking binding ofan IL-1-R intracellular ligand protein to the intracellular domain ofIL-1-R, and thus may act as inhibitors of IL-1-R intracellular domainbinding and/or IL-1 activity. Binding assays using a desired bindingprotein, immobilized or not, are well known in the art and may be usedfor this purpose using the IL-1-R intracellular ligand protein of theinvention. Examples 1 and 3 describe examples of such assays.Appropriate screening assays may be cell-based or cell-free.Alternatively, purified protein based screening assays may be used toidentify such agents. For example, IL-1-R intracellular ligand proteinmay be immobilized in purified form on a carrier and binding to purifiedIL-1-R intracellular domain may be measured in the presence and in theabsence of potential inhibiting agents. A suitable binding assay mayalternatively employ purified IL-1-R intracellular domain immobilized ona carrier, with a soluble form of a IL-1-R intracellular ligand proteinof the invention. Any IL-1-R intracellular ligand protein may be used inthe screening assays described above.

In such a screening assay, a first binding mixture is formed bycombining IL-1-R intracellular domain protein and IL-1-R intracellularligand protein, and the amount of binding in the first binding mixture(B_(o)) is measured. A second binding mixture is also formed bycombining IL-1-R intracellular domain protein. IL-1-R intracellularligand protein, and the compound or agent to be screened, and the amountof binding in the second binding mixture (B) is measured. The amounts ofbinding in the first and second binding mixtures are compared, forexample, by performing a B/B_(o) calculation. A compound or agent isconsidered to be capable of inhibiting IL-1-R intracellular domainbinding if a decrease in binding in the second binding mixture ascompared to the first binding mixture is observed. The formulation andoptimization of binding mixtures is within the level of skill in theart. Such binding mixtures may also contain buffers and salts necessaryto enhance or to optimize binding, and additional control assays may beincluded in the screening assay of the invention.

Alternatively, appropriate screening assays may be cell based. Forexample, the binding or interaction between an IL-1-R ligand protein andthe IL-1-R intracellular domain can be measured in yeast as describedbelow in Examples 1 and 3.

Compounds found to reduce, preferably by at least about 10% morepreferably greater than about 50% or more, the binding activity ofIL-1-R intracellular ligand protein to IL-1-R intracellular domain maythus be identified and then secondarily screened in other bindingassays, including in vivo assays. By these means compounds havinginhibitory activity for IL-1-R intracellular domain binding which may besuitable as anti-inflammatory agents may be identified.

Isolated IL-1-R intracellular ligand protein may be useful in treating,preventing or ameliorating inflammatory conditions and other conditionssuch as osteoporosis, colitis, myelogenous leukemia, diabetes, wastingand atherosclerosis. Isolated IL-1-R intracellular ligand protein may beused itself as an inhibitor of IL-1-R intracellular domain binding or todesign inhibitors of IL-1-R intracellular domain binding. Inhibitors ofbinding of IL-1-R intracellular ligand protein to the IL-1-Rintracellular domain ("IL-1-R intracellular binding inhibitors") arealso useful for treating such conditions.

The present invention encompasses both pharmaceutical compositions andtherapeutic methods of treatment or use which employ isolated IL-1-Rintracellular ligand protein and/or binding inhibitors of IL-1-Rintracellular binding.

Isolated IL-1-R intracellular ligand protein or binding inhibitors (fromwhatever source derived, including without limitation from recombinantand non-recombinant cell lines) may be used in a pharmaceuticalcomposition when combined with a pharmaceutically acceptable carrier.Such a composition may also contain (in addition to IL-1-R intracellularligand protein or binding inhibitor and a carrier) diluents, fillers,salts, buffers, stabilizers, solubilizers, and other materials wellknown in the art. The term "pharmaceutically acceptable" means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,G-CSF, Meg-CSF, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may further contain other anti-inflammatory agents. Suchadditional factors and/or agents may be included in the pharmaceuticalcomposition to produce a synergistic effect with isolated IL-1-Rintracellular ligand protein or binding inhibitor, or to minimize sideeffects caused by the isolated IL-1-R intracellular ligand protein orbinding inhibitor. Conversely, isolated IL-1-R intracellular ligandprotein or binding inhibitor may be included in formulations of theparticular cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent tominimize side effects of the cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent.

The pharmaceutical composition of the invention may be in the form of aliposome in which isolated IL-1-R intracellular ligand protein orbinding inhibitor is combined, in addition to other pharmaceuticallyacceptable carriers, with amphipathic agents such as lipids which existin aggregated form as micelles, insoluble monolayers, liquid crystals,or lamellar layers in aqueous solution. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871;U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323, all of which are incorporated herein by reference.

As used herein, the term "therapeutically effective amount" means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing prevention or amelioration of an inflammatoryresponse or condition, or an increase in rate of treatment, healing,prevention or amelioration of such conditions. When applied to anindividual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of isolated IL-1-R intracellular ligandprotein or binding inhibitor is administered to a mammal having acondition to be treated. Isolated IL-1-R intracellular ligand protein orbinding inhibitor may be administered in accordance with the method ofthe invention either alone or in combination with other therapies suchas treatments employing cytokines, lymphokines or other hematopoieticfactors. When co-administered with one or more cytokines, lymphokines orother hematopoietic factors, isolated IL-1-R intracellular ligandprotein or binding inhibitor may be administered either simultaneouslywith the cytokine(s), lymphokine(s), other hematopoietic factor(s),thrombolytic or anti-thrombotic factors, or sequentially. Ifadministered sequentially, the attending physician will decide on theappropriate sequence of administering isolated IL-1-R intracellularligand protein or binding inhibitor in combination with cytokine(s),lymphokine(s), other hematopoietic factor(s), thrombolytic oranti-thrombotic factors.

Administration of isolated IL-1-R intracellular ligand protein orbinding inhibitor used in the pharmaceutical composition or to practicethe method of the present invention can be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, or cutaneous,subcutaneous, or intravenous injection. Intravenous administration tothe patient is preferred.

When a therapeutically effective amount of isolated IL-1-R intracellularligand protein or binding inhibitor is administered orally, isolatedIL-1-R intracellular ligand protein or binding inhibitor will be in theform of a tablet, capsule, powder, solution or elixir. When administeredin tablet form, the pharmaceutical composition of the invention mayadditionally contain a solid carrier such as a gelatin or an adjuvant.The tablet, capsule, and powder contain from about 5 to 95% isolatedIL-1-R intracellular ligand protein or binding inhibitor, and preferablyfrom about 25 to 90% isolated IL-1-R intracellular ligand protein orbinding inhibitor. When administered in liquid form, a liquid carriersuch as water, petroleum, oils of animal or plant origin such as peanutoil, mineral oil, soybean oil, or sesame oil, or synthetic oils may beadded. The liquid form of the pharmaceutical composition may furthercontain physiological saline solution, dextrose or other saccharidesolution, or glycols such as ethylene glycol, propylene glycol orpolyethylene glycol. When administered in liquid form, thepharmaceutical composition contains from about 0.5 to 90% by weight ofisolated IL-1-R intracellular ligand protein or binding inhibitor, andpreferably from about 1 to 50% isolated IL-1-R intracellular ligandprotein or binding inhibitor.

When a therapeutically effective amount of isolated IL-1-R intracellularligand protein or binding inhibitor is administered by intravenous,cutaneous or subcutaneous injection, isolated IL-1-R intracellularligand protein or binding inhibitor will be in the form of apyrogen-free parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to isolated IL-1-Rintracellular ligand protein or binding inhibitor, an isotonic vehiclesuch as Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, Lactated Ringer'sInjection, or other vehicle as known in the art. The pharmaceuticalcomposition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives known to thoseof skill in the art.

The amount of isolated IL-1-R intracellular ligand protein or bindinginhibitor in the pharmaceutical composition of the present inventionwill depend upon the nature and severity of the condition being treated,and on the nature of prior treatments which the patient has undergone.Ultimately, the attending physician will decide the amount of isolatedIL-1-R intracellular ligand protein or binding inhibitor with which totreat each individual patient. Initially, the attending physician willadminister low doses of isolated IL-1-R intracellular ligand protein orbinding inhibitor and observe the patient's response. Larger doses ofisolated IL-1-R intracellular ligand protein or binding inhibitor may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 0.1 μgto about 100 mg of isolated IL-1-R intracellular ligand protein orbinding inhibitor per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the isolated IL-1-R intracellular ligandprotein or binding inhibitor will be in the range of 12 to 24 hours ofcontinuous intravenous administration. Ultimately the attendingphysician will decide on the appropriate duration of intravenous therapyusing the pharmaceutical composition of the present invention.

Isolated IL-1-R intracellular ligand protein of the invention may alsobe used to immunize animals to obtain polyclonal and monoclonalantibodies which specifically react with the IL-1-R intracellular ligandprotein and which may inhibit IL-1-R intracellular domain binding. Suchantibodies may be obtained using either the entire IL-1-R intracellularligand protein or fragments of IL-1-R intracellular ligand protein as animmunogen. The peptide immunogens additionally may contain a cysteineresidue at the carboxyl terminus, and are conjugated to a hapten such askeyhole limpet hemocyanin (KLH). Methods for synthesizing such peptidesare known in the art, for example, as in R. P. Merrifield, J.Amer.Chem.Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBSLett. 211, 10 (1987).

Monoclonal antibodies binding to IL-1-R intracellular ligand protein orto complex carbohydrate moieties characteristic of the IL-1-Rintracellular ligand glycoprotein may be useful diagnostic agents forthe immunodetection of IL-1-R ligand protein.

Neutralizing monoclonal antibodies binding to IL-1-R intracellularligand protein or to complex carbohydrates characteristic of IL-1-Rintracellular ligand glycoprotein may also be useful therapeutics forboth inflammatory conditions and also in the treatment of some forms ofcancer where abnormal expression of IL-1-R intracellular ligand proteinis involved. These neutralizing monoclonal antibodies are capable ofblocking the signaling function of the IL-1-R intracellular ligandprotein. By blocking the binding of IL-1-R intracellular ligand protein,certain biological responses to IL-1 are either abolished or markedlyreduced. In the case of cancerous cells or leukemic cells, neutralizingmonoclonal antibodies against IL-1-R intracellular ligand protein may beuseful in detecting and preventing the metastatic spread of thecancerous cells, which may be mediated by the IL-1-R intracellularligand protein.

Due to the similarity of its sequence to SEQ ID NO:6, CANP and fragmentsthereof which bind to the IL-1-R intracellular domain are proteinshaving IL-1-R intracellular ligand protein activity as defined herein.As a result, they are also useful in pharmaceutical compositions, fortreating inflammatory conditions and for inhibiting IL-1-R intracellulardomain binding as described above for IL-1-R intracellular ligandproteins generally.

EXAMPLE 1 CLONING OF IL-1-R INTRACELLULAR LIGAND PROTEIN ENCODINGPOLYNUCLEOTIDE

A yeast genetic selection method, the "interaction trap" [Gyuris et al,Cell 75:791-803, 1993, which is incorporated herein by reference], wasused to screen WI38 and HeLa cell cDNA libraries (preparation see below)for proteins that interact with IL-1-R-1c, the cytoplasmic portion(intracellular domain) of the interleukin-1 receptor p80, or type I. TheIL-1-R-1c DNA, encoding amino acids 340 to 552 of the type I IL-1receptor, was obtained via the polymerase chain reaction (PCR) of ahuman WI38 cell cDNA library. This IL-1-R-1c DNA was then cloned intopEG202 by an EcoRI site, generating the bait plasmid, pEG202-IL-1-R-1c.This plasmid contains the HIS3 selectable marker, and expression of thebait, the LexA-IL-1-R-1c fusion protein, is from the strong constitutiveADH1 promoter. To create the reporter strain carrying the bait protein,yeast strain EGY48, containing the reporter sequence LexAop-Leu2 inplace of the chromosomal LEU2, was transformed with pEG202-IL-1-R-1c andpSH18-34 (Ura+), which carries another reporter sequence, LexAop-lacZ.For screening cDNAs encoding proteins that interact with IL-1-R-1c, theexpression vector pJG4-5 (TRP1), containing either a WI38 or HeLa cellcDNA library (see below for the cDNA library construction), wastransformed into the above strain (EGY48/pEG202-IL-1-R-1c/pSH18-34)according to the method described by Gietz et al., Nucleic Acids Res.,20, 1425, 1992.

The bait used in obtaining clones 14w, 19w and 31w was constructed bycloning the DNA sequences encoding amino acids 477 to 527 of IL-1receptor p80 into the EcoRI and NotI sites of EG202. The resultingplasmid was named EG202-IL1R (477-527). This region of the IL-1 receptoris believed to be essential for signaling.

cDNA Library Construction:

W138 cell cDNA library: Double stranded cDNA was prepared from 3 μg ofWI38 mRNA using reagents provided by the Superscript Choice System(Gibco/BRL, Gaithersberg, Md.) with the following substitutions: thefirst strand synthesis was primed using an oligo dT/XhoI primer/linker,and the dNTP mix was substituted with a mix containing methyl dCTP(Stratagene, LaJolla, Calif.). The cDNA was modified at both ends byaddition of an EcoRI/NotI/SalI adapter linker and subsequently digestedwith XhoI. This produced cDNA molecules possessing an EcoRI/NotI/SalIoverhang at the 5' end of the gene and an XhoI overhang at the 3' end.These fragments were then ligated into the yeast expression/fusionvector pJG4-5 (Gyuris et al., Cell, 75, 791-803, 1993), which containsat its amino terminus, the influenza virus HA1 epitope tag, the B42acidic transcription activation domain, and the SV40 nuclearlocalization signal, all under the control of the galactose-dependentGAL1 promoter. The resulting plasmids were then electroporated intoDH10B cells (Gibco/BRL). A total of 7.1×10⁶ colonies were plated on LBplates containing 100 ug/ml of ampicillin. These E.coli were scraped,pooled, and a large scale plasmid prep was performed using the WizardMaxi Prep kit (Promega, Madison, Wis.), yielding 3.2 mg of supercoiledplasmid DNA.

HeLa cell cDNA: HeLa cell cDNA preparation methods are described inGyuris et al., Cell, 75, 791-803, 1993, which is incorporated herein byreference.

HeLa Cell CDNA Screening Results:

2×10⁵ transformants were obtained on glucose Ura⁻ His⁻ Trp³¹ plates.These transformants were pooled and resuspended in a solution of 65%glycerol, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂ and stored at -80° C. in1mL aliquots. For screening purposes, aliquots of these were diluted10-fold into Ura⁻ His⁻ Trp⁻ CM dropout gal/raff medium (containing 2%galactose, 1% raffinose), which induces the expresssion of the libraryencoded proteins, and incubated at 30° C. for 4 hours. 2×10⁶ colonyforming units (CFUs) were then plated on standard 10 cm galactose X-GalUra⁻ His⁻ Trp⁻ Leu⁻ plates at a density of 2×10⁵ CFU/plate. After 4 daysat 30° C., colonies that were strong LacZ⁺ were chosen for furtherprocessing. In order to test if the Leu⁺ /LacZ⁺ phenotype was due to thelibrary-encoded protein, the galactose dependency of the phenotype wastested. Expression of the library-encoded proteins was turned off bygrowth on glucose Ura⁻ His⁻ Trp⁻ master plates and then retested forgalactose-dependency on glucose Ura⁻ His⁻ Trp⁻ Leu⁻, galactose Ura⁻ His⁻Trp⁻ Leu⁻ glucose X-Gal Ura⁻ His⁻ Trp⁻, and galactose X-Gal Ura⁻ His⁻Trp⁻ plates. Of these many colonies showed galactose-dependent growth onLeu⁻ plates and galactose-dependent blue color on X-Gal-containingmedium (LacZ⁻ phenotype). Total yeast DNA was prepared from thesecolonies according to the method described previously (Hoffman andWinston, 1987). In order to analyze the cDNA sequences. PCR reactionswere performed using the above yeast DNA as a template and oligo primersspecific for the vector pJG4-5, flanlking the cDNA insertion point. PCRproducts were purified (Qiagen PCR purification kit), subjected torestriction digest with the enzyme HaeIII run on 1.8% agarose gels, andthe restriction patterns compared. Similar and identical restrictionpatterns were grouped and representatives of each group were sequencedand compared to Genbank and other databases to identify any sequencehomologies.

W138 Cell cDNA Screening Results:

This screen was performed as above with the following exceptions:1)1×10⁶ transformants were obtained on glucose Ura⁻ His⁻ Trp⁻ plates andpooled. 2) 11×10⁶ CFU were screened. Of these, 0.5% were Leu⁺ and ofthose, 1% were LacZ₊. This gave a frequency of 50 double positives per10⁶ transformants screened. Colonies, exhibiting a strong LacZ⁺phenotype (as judged by the strength of blue color on X-Gal containingmedium), were chosen for further processing. Clones with the strongestLacZ+ phenotype were chosen for further specificity tests as describedabove.

A WI38 cDNA library was transformed into the reporter strain(EGY48/pSH18-34) containing the bait plasmid EG202-IL1R (477-527). 1.3million primary transformants were harvested and 7 million colonies werescreened. 192 galactose-dependent colonies were isolated. Among these,51 clones were bait specific (i.e., interacted specifically with theoriginal bait, but not with an unrelated bait, bicoid). These cloneswere then subjected to DNA sequence analysis. Clones 19w was isolated 6,times, clone 31w twice, and clone 14w once.

EXAMPLE 2 EXPRESSION OF THE IL-1-R INTRACELLULAR LIGAND PROTEIN

cDNAs encoding IL-1-R intracellular ligand proteins were released fromthe pJG4-5 vector with the appropriate restriction enzymes. For example,EcoRI and XhoI were used to release cDNA from the relevant clone. Wherethe restriction sites were also present in the internal sequence of thecDNA, PCR was performed to obtain the cDNA. These cDNAs were then clonedinto various expression vectors. These included pGEX (Pharmacia) or pMAL(New England Biolabs) for expression as a GST(Glutathione-S-transferase) or MBP (maltose binding protein) fusionprotein in E. coli, a pED-based vector for mammalian expression, and pVLor pBlueBacHis (Invitrogen) for baculovirus/insect expression. For theimmunodetection of IL-1-R intracellular ligand expression in mammaliancells, an epitope sequence, "Flag," was inserted into the translationalstart site of the pED vector, generating the pED-Flag vector. cDNAs werethen inserted into the pED-Flag vector. Thus, the expression of cDNAfrom pED-Flag yields a protein with an amino terminal Met, followed bythe "Flag" sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. StandardDEAE-Dextran or lipofectamine methods were used to transfect COS or CHOdukx cells. Immunodetection of Flag-tagged proteins was achieved usingthe M2 antibody (Kodak). Moreover, an immunoaffinity column using the M2antibody, followed by elution with the "Flag" peptide, can be used forthe rapid purification of the flag-tagged protein. Similarly, affinitypurification of GST-, MBP- or His-tagged fusion proteins can beperformed using glutathione, amylose, or nickel columns. Detailedpurification protocols are provided by the manufacturers. For manyfusion proteins, the IL-1-R intracellular ligand can be released by theaction of thrombin, factor Xa, or enterokinase cleavage. In the casewhere highly purified material is required, standard purificationprocedures, such as ion-exchange, hydrophobic, and gel filtrationchromatography will be applied in addition to the affinity purificationstep.

FIG. 1 depicts an autoradiograph demonstrating the expression of IL-1-Rintracellular ligand proteins in mammalian cells. FIG. 1 shows theresults of expression of Flag-14w, -19w and -31w in COS cells. COS cellswere transfected with either pED-Flag (vector control), Flag-14w, -19wor -31w plasmid by the lipofectamine method. Thirty μg of each celllysate were prepared and subjected to 4-20% SDS gel electrophoresis,followed by Western blot analysis using M2 antibody (Kodak). AFlag-containing protein, Flag-BAP (Kodak), was also loaded as astandard. The bands in the Flag-14w, -19w and -31w indicate significantexpression of the respective IL-1-R intracellular ligand proteins.

EXAMPLE 3 ASSAYS OF IL-1-R INTRACELLULAR DOMAIN BINDING

Two different methods were used to assay for IL-1-R intracellular ligandprotein activity. The first assay measures binding in the yeast strainin "interaction trap," the system used here to screen for IL-1-R-1cinteracting proteins. In this system, the expression of reporter genesfrom both LexAop-Leu2 and LexAop-LacZ relies on the interaction betweenthe bait protein, in this case IL-1-R-1c, and the prey, the IL-1-Rintracellular ligand. Thus, one can measure the strength of theinteraction by the level of Leu2 or LacZ expression. The most simplemethod is to measure the activity of the LacZ encoded protein,β-galactosidase. This activity can be judged by the degree of bluenesson the X-Gal containing medium or filter. For the quantitativemeasurement of β-galactosidase activity, standard assays can be found in"Methods in Yeast Genetics" Cold Spring Harbor, N.Y., 1990 (by Rose, M.D., Winston, F., and Hieter, P.).

The second assay for measuring binding is a cell-free system. An exampleof a typical assay is described below. Purified MBP-IL-1-R-1c fusionprotein (2 μg) was mixed with glutathione-Sepharose 4 B beads bound witha GST-IL-1-R-1c intracellular ligand for 2 hour at 4° C. The mixture wasthen centrifuged to separate bound (remained with the beads) and unbound(remained in the supernatant) MBP-IL-1-R-1c. After extensive washing,the bound MBP-IL-1-R-1c was eluted with glutathione and detected byWestern blot analysis using an MBP antibody. The IL-1-R-1c or theintracellular ligand can also be immobilized on other solid supports,such as on plates or fluorobeads. The binding can then be measured usingELISA or SPA (scintillation proximity assay).

EXAMPLE 4 CHARACTERIZATION OF IL-1-R INTRACELLULAR LIGAND PROTEIN

Mapping the interaction site in IL-1-R-1c

Many of the key amino acids for IL-1-R signaling have been determined bysite-directed mutagenesis (Heguy et al., 1992, JBC, 267, 2605-2609).These amino acids are conserved between IL-1-R and the Drosophila Tollprotein, which is required for transducing dorsoventral positionalinformation to cells in the developing embryo. In order to test if theIL-1-R intracellular proteins interact with these residues, theseresidues were mutagenized and the ability of the mutant protein tointeract with the intracellular ligand in the "interaction trap" systemwas tested. Mutations that abolish IL-1R signaling were introduced intothe original bait plasmid, EG202-IL-1R (477-527) (with-following aminoacid substitutions: F513A, W514A, K515R, R518K, and Y519S) and theability of the IL-1R intracellular ligands to interact with these mutantproteins was tested in the interaction trap. EGY48 carrying pSH18-34(lexAop-LacZ) were cotransformed with two plasmids: one carrying 14w,19w or 31w; the other with bait, EG202-IL1R (477-527), either wild-typeor one of the mutants. transformants were then streaked onto CM ura⁻his⁻ trp⁻ plates containing galactose/raffinose and β-gal. The strengthof interaction (as indicated by the number of "+" signs) was judged bythe blueness in the plates (indicator of LacZ expression). The resultsare summarized in Table I.

                  TABLE I                                                         ______________________________________                                        bait →                                                                       WT      F513A   W514A  K515R  R518K Y519S                                 clone ↓                                                              ______________________________________                                        14w   +++     ++      +      +++    +++   +                                     19w + - + ++ - -                                                              31w ++ - - +++ - -                                                          ______________________________________                                    

Clone 14w interacted with mutant baits W514A and Y519S much more weaklythan with wild-type bait. Clone 19w interacted differentially withwild-type and manu of the mutant baits. It appeared to interact with themutant bait K515R more strongly than with wild-type, while reducedinteraction was observed with mutant baits F513R, R518K and Y519S. Theinteraction of clone 31w was significantly reduced by mutations F513A,W514A, R518K and Y519S. The change in the interaction strength by thesemutations suggests that these residues are the site(s) of interaction.therefore, these data suggest that clones 14w, 19w and 31w interact withmany of the signaling residues and may play a role in IL-1R signaling.

Effect on the IL-1-mediated response

The effect of the IL-1-R intracellular ligands on the IL-1-mediatedresponse can be evaluated in cells overexpressing the ligands. A numberof IL-1 mediated responses, including transient or prolonged responses,can be measured. For example, IL-1-induced kinase activity toward eitherMBP (myelin basic protein) or the N-terminus (amino acids 1-79) of c-juncan be measured in COS cells or CHO cells either transiently or stablyoverexpressing IL-1R intracellular ligand proteins. Alternatively, otherfunctional assays, such as the induction of gene expression or PGE₂production after prolonged incubation with IL-1, can also be used tomeasure the IL-1 mediated response. Conversely, the significance of theIL-1-R intracellular ligand proteins in IL-1signaling can be establishedby lowering or eliminating the expression of the ligands. Theseexperiments can be performed using antisense expression or transgenicmice.

IL-1 mediated JNK (c-jun NH₂ -terminal kinase, Derjard et al., Cell1994, 76, 1025-1037) activation was used to study the effect of theIL-1R intracellular ligands on IL-1 signaling. COS cells weretransfected with both pEDflag plasmid containing one of the clones(e.g., 19w) and HA-JNK1 plasmid by the DEAE-dextran method. 48 hrs aftertransfection, cells were starved in 0.1% BSA for 1 hr and treated withvarious amounts of IL-1α for 15 min. Cells were then lysed, centrifugedand immunoprecipitated with anti-HA monoclonal antibody, 12CA5(Boehringer Mannheim). JNK activity was performed at 30° C. for 20 minusing 5 μg GST-c-jun (1-79 amino acids), 20 μM ATP, and 5 μCi [γ-³²P]ATP in 40 μl of kinase buffer (25 mM HEPES, pH 7.5, 20 mM MgCl₂, 20 mMμ-glycerophosphate, 0.1 mM sodium orthovanadate, 2 mM DTT). Thereactions were terminated using laemmli sample buffer and the productswere resolved by SDS-PAGE (4-20%).

As shown in FIG. 2, expression of clone 19w stimulated JNK activity inall IL-1 concentrations tested as compared to the pED flag vectortransfected cells. It also enhanced JNK activity even in the absence ofIL-1. These data strongly suggest that clone 19w, through itsinteraction with the signaling domain of IL-1 receptor (i.e., aminoacids 477-527 of IL-1R), may indeed participate in the signaling event.

Enzymatic or functional assays

The signal transduction events initiated by IL-1 binding to its receptorare still largely unknown. However, one major result of IL-1 binding isthe stimulation of cellular serine/threonine kinase activity. Inaddition, IL-1 has been shown to stimulate the activity of PC-PLC, PLA₂,and sphingomyelinase. Therefore, some of the IL-1-R intracellular ligandproteins may possess intrinsic enzymatic activity that is responsiblefor these activities. Therefore, enzymatic assays can be performed totest this possibility, particularly with those clones that encodeproteins with sequence homology to known enzymes. In addition toenzymatic activity, based o n the sequence homology to proteins w it hknown function, other functional assays, for instance, ATPbinding/transporter activity for the full length protein of clone 140,can also be measured.

EXAMPLE 5 ISOLATION OF FULL LENGTH CLONES

In many cases, cDNAs obtained from the interaction trap method eachencode only a portion of the full length protein. Therefore, it isdesirable to isolate full length clones. The cDNAs obtained from thescreening are used as probes, and the cDNA libraries described herein,or alternatively phage cDNA libraries, are screened to obtain fulllength clones in accordance with known methods (see for example,"Molecular Cloning, A Laboratory Manual", by Sambrook et al., 1989 ColdSpring Harbor).

EXAMPLE 6 ANTIBODIES SPECIFIC FOR IL-1-R INTRACELLULAR LIGAND PROTEIN

Antibodies specific for IL-1-R intracellular ligand proteins can beproduced using purified recombinant protein, as described in Example 2,as antigen. Both polyclonal and monoclonal antibodies will be producedusing standard techniques, such as those described in "Antibodies, aLaboratory Manual" by Ed Harlow and David Lane (1988), Cold Spring,Harbor Laboratory.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 7                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1571 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..529                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - G ATC CCC AGG GTG GAC CTC CGG GTG TGG CAG - #GAC TGC TGT GAA GAC             46                                                                         Ile Pro Arg Val Asp Leu Arg Val Trp G - #ln Asp Cys Cys Glu Asp                 1              - # 5                 - # 10                 - # 15         - - TGT AGG ACC AGG GGG CAG TTC AAT GCC TTT TC - #C TAT CAT TTC CGA GGC           94                                                                       Cys Arg Thr Arg Gly Gln Phe Asn Ala Phe Se - #r Tyr His Phe Arg Gly                            20 - #                 25 - #                 30              - - AGA CGG TCT CTT GAG TTC AGC TAC CAG GAG GA - #C AAG CCG ACC AAG AAA          142                                                                       Arg Arg Ser Leu Glu Phe Ser Tyr Gln Glu As - #p Lys Pro Thr Lys Lys                        35     - #             40     - #             45                  - - ACA AGA CCA CGG AAA ATA CCC AGT GTT GGG AG - #A CAG GGG GAA CAT CTC          190                                                                       Thr Arg Pro Arg Lys Ile Pro Ser Val Gly Ar - #g Gln Gly Glu His Leu                    50         - #         55         - #         60                      - - AGC AAC AGC ACC TCA GCC TTC AGC ACA CGC TC - #A GAT GCA TCT GGG ACA          238                                                                       Ser Asn Ser Thr Ser Ala Phe Ser Thr Arg Se - #r Asp Ala Ser Gly Thr                65             - #     70             - #     75                          - - AAT GAC TTC AGA GAG TTT GTT CTG GAA ATG CA - #G AAG ACC ATC ACA GAC          286                                                                       Asn Asp Phe Arg Glu Phe Val Leu Glu Met Gl - #n Lys Thr Ile Thr Asp            80                 - # 85                 - # 90                 - # 95       - - CTC AGA ACA CAG ATA AAG AAA CTT GAA TCA CG - #G CTC AGT ACC ACA GAG          334                                                                       Leu Arg Thr Gln Ile Lys Lys Leu Glu Ser Ar - #g Leu Ser Thr Thr Glu                           100  - #               105  - #               110              - - TGC GTG GAT GCC GGG GGC GAA TCT CAC GCC AA - #C AAC ACC AAG TGG AAA          382                                                                       Cys Val Asp Ala Gly Gly Glu Ser His Ala As - #n Asn Thr Lys Trp Lys                       115      - #           120      - #           125                  - - AAA GAT GCA TGC ACC ATT TGT GAA TGC AAA GA - #C GGG CAG GTC ACC TGC          430                                                                       Lys Asp Ala Cys Thr Ile Cys Glu Cys Lys As - #p Gly Gln Val Thr Cys                   130          - #       135          - #       140                      - - TTC GTG GAA GCT TGC CCC CCT GCC ACC TGT GC - #T GTC CCC GTG AAC ATC          478                                                                       Phe Val Glu Ala Cys Pro Pro Ala Thr Cys Al - #a Val Pro Val Asn Ile               145              - #   150              - #   155                          - - CCA GGG GCC TGC TGT CCA GTC TGC TTA CAG AA - #G AGG GCG GAG GAA AAG          526                                                                       Pro Gly Ala Cys Cys Pro Val Cys Leu Gln Ly - #s Arg Ala Glu Glu Lys           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - CCC TAGGCTCCTG GGAGGCTCCT CAGAGTTTGT CTGCTGTGCC ATCGTGAGA - #T               579                                                                      Pro                                                                            - - CGGGTGGCCG ATGGCAGGGA GCTGCGGACT GCAGACCAGG AAACACCCAG AA -             #CTCGTGAC    639                                                                 - - ATTTCATGAC AACGTCCAGC TGGTGCTGTT ACAGAAGGCA GTGCAGGAGG CT -            #TCCAACCA    699                                                                 - - GAGCATCTGC GGAGAAGGAG GCACAGCAGG TGCCTGAAGG GAAGCAGGCA GG -            #AGTCCTAG    759                                                                 - - CTTCACGTTA GACTTCTCAG GTTTTTATTT AATTCTTTTA AAATGAAAAA TT -            #GGTGCTAC    819                                                                 - - TATTAAATTG CACAGTTGAA TCATTTAGGC GCCTAAATTG ATTTTGCCTC CC -            #AACACCAT    879                                                                 - - TTCTTTTTAA ATAAAGCAGG ATACCTCTAT ATGTCAGCCT TGCCTTGTTC AG -            #ATGCCAGG    939                                                                 - - AGCCGGCAGA CCTGTCACCC GCAGGTGGGG TGAGTCTCGG AGCTGCCAGA GG -            #GGCTCACC    999                                                                 - - GAAATCGGGG TTCCATCACA AGCTATGTTT AAAAAGAAAA TTGGTGTTTG CC -            #AAACGGAA   1059                                                                 - - CAGAACCTTT GATGAGAGCG TTCACAGGGA CACTGTCTGG GGGTGCAGTG CA -            #AGCCCCCG   1119                                                                 - - GCCTCTTCCC TGGGAACCTC TGAACTCCTC CTTCCTCTGG GCTCTCTGTA AC -            #ATTTCACC   1179                                                                 - - ACACGTCAGC ATCTAATCCC AAGACAAACA TTCCCGCTGC TCGAAGCAGC TG -            #TATAGCCT   1239                                                                 - - GTGACTCTCC GTGTGTCAGC TCCTTCCACA CCTGATTAGA ACATTCATAA GC -            #CACATTTA   1299                                                                 - - GAAACAGGTT TGCTTTCAGC TGTCACTTGC ACACATACTG CCTAGTTGTG AA -            #CCAAATGT   1359                                                                 - - GAAAAAACCT CCTTCATCCC ATTGTGTATC TGATACCTGC CGAGGGCCAA GG -            #GTGTGTGT   1419                                                                 - - TGACAACGCC GCTCCCAGCC GGCCCTGGTT GCGTCCACGT CCTGAACAAG AG -            #CCGCTTCC   1479                                                                 - - GGATGGCTCT TCCCAAGGGA GGAGGAGCTC AAGTGTCGGG AACTGTCTAA CT -            #TCAGGTTG   1539                                                                 - - TGTGAGTGCG TTAAAAAAAA AAAAAAAAAA AA       - #                  - #            1571                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 176 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Ile Pro Arg Val Asp Leu Arg Val Trp Gln As - #p Cys Cys Glu Asp Cys        1               5 - #                 10 - #                 15              - - Arg Thr Arg Gly Gln Phe Asn Ala Phe Ser Ty - #r His Phe Arg Gly Arg                   20     - #             25     - #             30                  - - Arg Ser Leu Glu Phe Ser Tyr Gln Glu Asp Ly - #s Pro Thr Lys Lys Thr               35         - #         40         - #         45                      - - Arg Pro Arg Lys Ile Pro Ser Val Gly Arg Gl - #n Gly Glu His Leu Ser           50             - #     55             - #     60                          - - Asn Ser Thr Ser Ala Phe Ser Thr Arg Ser As - #p Ala Ser Gly Thr Asn       65                 - # 70                 - # 75                 - # 80       - - Asp Phe Arg Glu Phe Val Leu Glu Met Gln Ly - #s Thr Ile Thr Asp Leu                       85 - #                 90 - #                 95              - - Arg Thr Gln Ile Lys Lys Leu Glu Ser Arg Le - #u Ser Thr Thr Glu Cys                  100      - #           105      - #           110                  - - Val Asp Ala Gly Gly Glu Ser His Ala Asn As - #n Thr Lys Trp Lys Lys              115          - #       120          - #       125                      - - Asp Ala Cys Thr Ile Cys Glu Cys Lys Asp Gl - #y Gln Val Thr Cys Phe          130              - #   135              - #   140                          - - Val Glu Ala Cys Pro Pro Ala Thr Cys Ala Va - #l Pro Val Asn Ile Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Ala Cys Cys Pro Val Cys Leu Gln Lys Ar - #g Ala Glu Glu Lys        Pro                                                                                             165  - #               170  - #               175             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1088 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..961                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - G AAA AAA GGA GGT AAA ACA GAA CAG GAT GGC - #TAT CAG AAA CCC ACC             46                                                                          Lys Lys Gly Gly Lys Thr Glu Gln Asp G - #ly Tyr Gln Lys Pro Thr                 1              - # 5                 - # 10                 - # 15         - - AAC AAA CAC TTC ACG CAG AGT CCC AAG AAG TC - #A GTG GCC GAC CTG CTG           94                                                                       Asn Lys His Phe Thr Gln Ser Pro Lys Lys Se - #r Val Ala Asp Leu Leu                            20 - #                 25 - #                 30              - - GGG TCC TTT GAA GGC AAA CGA AGA CTC CTT CT - #G ATC ACT GCT CCC AAG          142                                                                       Gly Ser Phe Glu Gly Lys Arg Arg Leu Leu Le - #u Ile Thr Ala Pro Lys                        35     - #             40     - #             45                  - - GCT GAG AAC AAT ATG TAT GTG CAA CAA CGT GA - #T GAA TAT CTG GAA AGT          190                                                                       Ala Glu Asn Asn Met Tyr Val Gln Gln Arg As - #p Glu Tyr Leu Glu Ser                    50         - #         55         - #         60                      - - TTC TGC AAG ATG GCT ACC AGG AAA ATC TCT GT - #G ATC ACC ATC TTC GGC          238                                                                       Phe Cys Lys Met Ala Thr Arg Lys Ile Ser Va - #l Ile Thr Ile Phe Gly                65             - #     70             - #     75                          - - CCT GTC AAC AAC AGC ACC ATG AAA ATC GAC CA - #C TTT CAG CTA GAT AAT          286                                                                       Pro Val Asn Asn Ser Thr Met Lys Ile Asp Hi - #s Phe Gln Leu Asp Asn            80                 - # 85                 - # 90                 - # 95       - - GAG AAG CCC ATG CGA GTG GTG GAT GAT GAA GA - #C TTG GTA GAC CAG CGT          334                                                                       Glu Lys Pro Met Arg Val Val Asp Asp Glu As - #p Leu Val Asp Gln Arg                           100  - #               105  - #               110              - - CTC ATC AGC GAG CTG AGG AAA GAG TAC GGA AT - #G ACC TAC AAT GAC TTC          382                                                                       Leu Ile Ser Glu Leu Arg Lys Glu Tyr Gly Me - #t Thr Tyr Asn Asp Phe                       115      - #           120      - #           125                  - - TTC ATG GTG CTA ACA GAT GTG GAT CTG AGA GT - #C AAG CAA TAC TAT GAG          430                                                                       Phe Met Val Leu Thr Asp Val Asp Leu Arg Va - #l Lys Gln Tyr Tyr Glu                   130          - #       135          - #       140                      - - GTA CCA ATA ACA ATG AAG TCT GTG TTT GAT CT - #G ATC GAT ACT TTC CAG          478                                                                       Val Pro Ile Thr Met Lys Ser Val Phe Asp Le - #u Ile Asp Thr Phe Gln               145              - #   150              - #   155                          - - TCC CGA ATC AAA GAT ATG GAG AAG CAG AAG AA - #G GAG GGC ATT GTT TGC          526                                                                       Ser Arg Ile Lys Asp Met Glu Lys Gln Lys Ly - #s Glu Gly Ile Val Cys           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - AAA GAG GAA GTT GGG GGA GTG TTA GAA CTG TT - #C CCA ATT AAT GGG        AGC      574                                                                    Lys Glu Glu Val Gly Gly Val Leu Glu Leu Ph - #e Pro Ile Asn Gly Ser                          180  - #               185  - #               190              - - TCT GTT GTT GAG CGA GAA GAC GTA CCA GCC CA - #T TTG GTG AAA GAC ATT          622                                                                       Ser Val Val Glu Arg Glu Asp Val Pro Ala Hi - #s Leu Val Lys Asp Ile                       195      - #           200      - #           205                  - - CGT AAC TAT TTT CAA GTG AGC CCG GAG TAC TT - #C TCC ATG CTT CTA GTC          670                                                                       Arg Asn Tyr Phe Gln Val Ser Pro Glu Tyr Ph - #e Ser Met Leu Leu Val                   210          - #       215          - #       220                      - - GGA AAA GAC GGA AAT GTC AAA TCC TGG TAT CC - #T TCC CCA ATG TGG TCC          718                                                                       Gly Lys Asp Gly Asn Val Lys Ser Trp Tyr Pr - #o Ser Pro Met Trp Ser               225              - #   230              - #   235                          - - ATG GTG ATT GTG TAC GAT TTA ATT GAT TCG AT - #G CAA CTT CGG AGA CAG          766                                                                       Met Val Ile Val Tyr Asp Leu Ile Asp Ser Me - #t Gln Leu Arg Arg Gln           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - GAA ATG GCG ATT CAG CAG TCA CTG GGG ATG CG - #C TGC CAG AAG ATG        AGT      814                                                                    Glu Met Ala Ile Gln Gln Ser Leu Gly Met Ar - #g Cys Gln Lys Met Ser                          260  - #               265  - #               270              - - ATG CAG GCT ATG GTT ACC ATA GTT ACC ACC AA - #G GAT ACC AGG ATG GTT          862                                                                       Met Gln Ala Met Val Thr Ile Val Thr Thr Ly - #s Asp Thr Arg Met Val                       275      - #           280      - #           285                  - - ACC AGG ATG ACT ACC GTC ATC ATG AGA GTT AT - #C ACC ATG GAT ACC CTT          910                                                                       Thr Arg Met Thr Thr Val Ile Met Arg Val Il - #e Thr Met Asp Thr Leu                   290          - #       295          - #       300                      - - ACT GAG CAG AAA TAT GTA ACC TTA GAC TCA GC - #C AGT TTC CTC TGC AGC          958                                                                       Thr Glu Gln Lys Tyr Val Thr Leu Asp Ser Al - #a Ser Phe Leu Cys Ser               305              - #   310              - #   315                          - - TGC TAAAACTACA TGTGGCCAGC TCCATTCTTC CACACTGCGT ACTACATTT - #C              1011                                                                       Cys                                                                           320                                                                            - - CTGCCTTTTT CTTTCAGTGT TTTTCTAAGA CTAAATAAAT AGCAAACTTT CA -             #CCTAAAAA   1071                                                                 - - AAAAAAAAAA AAAAAAA             - #                  - #                      - # 1088                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 320 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Lys Lys Gly Gly Lys Thr Glu Gln Asp Gly Ty - #r Gln Lys Pro Thr Asn        1               5 - #                 10 - #                 15              - - Lys His Phe Thr Gln Ser Pro Lys Lys Ser Va - #l Ala Asp Leu Leu Gly                   20     - #             25     - #             30                  - - Ser Phe Glu Gly Lys Arg Arg Leu Leu Leu Il - #e Thr Ala Pro Lys Ala               35         - #         40         - #         45                      - - Glu Asn Asn Met Tyr Val Gln Gln Arg Asp Gl - #u Tyr Leu Glu Ser Phe           50             - #     55             - #     60                          - - Cys Lys Met Ala Thr Arg Lys Ile Ser Val Il - #e Thr Ile Phe Gly Pro       65                 - # 70                 - # 75                 - # 80       - - Val Asn Asn Ser Thr Met Lys Ile Asp His Ph - #e Gln Leu Asp Asn Glu                       85 - #                 90 - #                 95              - - Lys Pro Met Arg Val Val Asp Asp Glu Asp Le - #u Val Asp Gln Arg Leu                  100      - #           105      - #           110                  - - Ile Ser Glu Leu Arg Lys Glu Tyr Gly Met Th - #r Tyr Asn Asp Phe Phe              115          - #       120          - #       125                      - - Met Val Leu Thr Asp Val Asp Leu Arg Val Ly - #s Gln Tyr Tyr Glu Val          130              - #   135              - #   140                          - - Pro Ile Thr Met Lys Ser Val Phe Asp Leu Il - #e Asp Thr Phe Gln Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Ile Lys Asp Met Glu Lys Gln Lys Lys Gl - #u Gly Ile Val Cys        Lys                                                                                             165  - #               170  - #               175             - - Glu Glu Val Gly Gly Val Leu Glu Leu Phe Pr - #o Ile Asn Gly Ser Ser                  180      - #           185      - #           190                  - - Val Val Glu Arg Glu Asp Val Pro Ala His Le - #u Val Lys Asp Ile Arg              195          - #       200          - #       205                      - - Asn Tyr Phe Gln Val Ser Pro Glu Tyr Phe Se - #r Met Leu Leu Val Gly          210              - #   215              - #   220                          - - Lys Asp Gly Asn Val Lys Ser Trp Tyr Pro Se - #r Pro Met Trp Ser Met      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Ile Val Tyr Asp Leu Ile Asp Ser Met Gl - #n Leu Arg Arg Gln        Glu                                                                                             245  - #               250  - #               255             - - Met Ala Ile Gln Gln Ser Leu Gly Met Arg Cy - #s Gln Lys Met Ser Met                  260      - #           265      - #           270                  - - Gln Ala Met Val Thr Ile Val Thr Thr Lys As - #p Thr Arg Met Val Thr              275          - #       280          - #       285                      - - Arg Met Thr Thr Val Ile Met Arg Val Ile Th - #r Met Asp Thr Leu Thr          290              - #   295              - #   300                          - - Glu Gln Lys Tyr Val Thr Leu Asp Ser Ala Se - #r Phe Leu Cys Ser Cys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1759 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..754                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - C AAA AAC TTC TTC CTG ACG AAT CGC GCC AGG - #GAG CGC TCA GAC ACC             46                                                                         Lys Asn Phe Phe Leu Thr Asn Arg Ala A - #rg Glu Arg Ser Asp Thr                 1              - # 5                 - # 10                 - # 15         - - TTC ATC AAC CTC CGG GAG GTG CTC AAC CGC TT - #C AAG CTG CCG CCA GGA           94                                                                       Phe Ile Asn Leu Arg Glu Val Leu Asn Arg Ph - #e Lys Leu Pro Pro Gly                            20 - #                 25 - #                 30              - - GAG TAC ATT CTC GTG CCT TCC ACC TTC GAA CC - #C AAC AAG GAT GGG GAT          142                                                                       Glu Tyr Ile Leu Val Pro Ser Thr Phe Glu Pr - #o Asn Lys Asp Gly Asp                        35     - #             40     - #             45                  - - TTC TGC ATC CGG GTC TTT TCT GAA AAG AAA GC - #T GAC TAC CAA GCT GTC          190                                                                       Phe Cys Ile Arg Val Phe Ser Glu Lys Lys Al - #a Asp Tyr Gln Ala Val                    50         - #         55         - #         60                      - - GAT GAT GAA ATC GAG GCC AAT CTT GAA GAG TT - #C GAC ATC AGC GAG GAT          238                                                                       Asp Asp Glu Ile Glu Ala Asn Leu Glu Glu Ph - #e Asp Ile Ser Glu Asp                65             - #     70             - #     75                          - - GAC ATT GAT GAT GGA TTC AGG AGA CTG TTT GC - #C CAG TTG GCA GGA GAG          286                                                                       Asp Ile Asp Asp Gly Phe Arg Arg Leu Phe Al - #a Gln Leu Ala Gly Glu            80                 - # 85                 - # 90                 - # 95       - - GAT GCG GAG ATC TCT GCC TTT GAG CTG CAG AC - #C ATC CTG AGA AGG GTT          334                                                                       Asp Ala Glu Ile Ser Ala Phe Glu Leu Gln Th - #r Ile Leu Arg Arg Val                           100  - #               105  - #               110              - - CTA GCA AAG CGC CAA GAT ATC AAG TCA GAT GG - #C TTC AGC ATC GAG ACA          382                                                                       Leu Ala Lys Arg Gln Asp Ile Lys Ser Asp Gl - #y Phe Ser Ile Glu Thr                       115      - #           120      - #           125                  - - TGC AAA ATT ATG GTT GAC ATG CTA GAT TCG GA - #C GGG AGT GGC AAG CTG          430                                                                       Cys Lys Ile Met Val Asp Met Leu Asp Ser As - #p Gly Ser Gly Lys Leu                   130          - #       135          - #       140                      - - GGG CTG AAG GAG TTC TAC ATT CTC TGG ACG AA - #G ATT CAA AAA TAC CAA          478                                                                       Gly Leu Lys Glu Phe Tyr Ile Leu Trp Thr Ly - #s Ile Gln Lys Tyr Gln               145              - #   150              - #   155                          - - AAA ATT TAC CGA GAA ATC GAC GTT GAC AGG TC - #T GGT ACC ATG AAT TCC          526                                                                       Lys Ile Tyr Arg Glu Ile Asp Val Asp Arg Se - #r Gly Thr Met Asn Ser           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - TAT GAA ATG CGG AAG GCA TTA GAA GAA GCA GG - #T TTC AAG ATG CCC        TGT      574                                                                    Tyr Glu Met Arg Lys Ala Leu Glu Glu Ala Gl - #y Phe Lys Met Pro Cys                          180  - #               185  - #               190              - - CAA CTC CAC CAA GTC ATC GTT GCT CGG TTT GC - #A GAT GAC CAG CTC ATC          622                                                                       Gln Leu His Gln Val Ile Val Ala Arg Phe Al - #a Asp Asp Gln Leu Ile                       195      - #           200      - #           205                  - - ATC GAT TTT GAT AAT TTT GTT CGG TGT TTG GT - #T CGG CTG GAA ACG CTA          670                                                                       Ile Asp Phe Asp Asn Phe Val Arg Cys Leu Va - #l Arg Leu Glu Thr Leu                   210          - #       215          - #       220                      - - TTC AAG ATA TTT AAG CAG CTG GAT CCC GAG AA - #T ACT GGA ACA ATA GAG          718                                                                       Phe Lys Ile Phe Lys Gln Leu Asp Pro Glu As - #n Thr Gly Thr Ile Glu               225              - #   230              - #   235                          - - CTC GAC CTT ATC TCT TGG CTC TGT TTC TCA GT - #A CTT TGAAGTTATA               764                                                                       Leu Asp Leu Ile Ser Trp Leu Cys Phe Ser Va - #l Leu                           240                 2 - #45                 2 - #50                            - - ACTAATCTGC CTGAAGACTT CTCATGATGG AAAATCAGCC AAGGACTAAG CT -             #TCCATAGA    824                                                                 - - AATACACTTT GTATCTGGAC CTCAAAATTA TGGGAACATT TACTTAAACG GA -            #TGATCATA    884                                                                 - - GCTGAAAATA ATGATACTGT CAATTTGAGA TAGCAGAAGT TTCACACATC AA -            #AGTAAAAG    944                                                                 - - ATTTGCATAT CATTATACTA AATGCAAATG AGTCGCTTAA CCCTTGACAA GG -            #TCAAAGAA   1004                                                                 - - AGCTTTAAAT CTGTAAATAG TATACACTTT TTACTTTTAC ACACTTTCCT GT -            #TCATAGCA   1064                                                                 - - ATATTAAATC AGGAAAAAAA AATGCAGGGA GGTATTTAAC AGCTGAGCAA AA -            #ACATTGAG   1124                                                                 - - TCGCTCTCAA AGGACACGAG GCCCTTGGCA GGGAATATTT AAAGCAACTT CA -            #AGTTTAAA   1184                                                                 - - ATGCAGCTGT TGATTCTACC AAACAACAGT CCAAGATTAC CATTTCCCAT GA -            #GCCAACTG   1244                                                                 - - GGAAACATGG TATATCATGA AGTAATCTTG TCAAGGCATC TGGAGAGTCC AG -            #GAGAGAAG   1304                                                                 - - ACTCACCTCT GTCGCTTGGG TTAAACAAGA GACAGGTTTT GTAGAATATT GA -            #TTGGTAAT   1364                                                                 - - AGTAAATCGT TCTCCTTACA ATCAAGTTCT TGACCCTATT CGGCCTTATA CA -            #TCTGGTCT   1424                                                                 - - TACAAAGACC AAAGGGATCC TGCGCTTGAT CAACTGAACC AGTATGCCAA AA -            #CCAGGCAT   1484                                                                 - - CCAATTTGTA AACCAATTAT GATAAAGGAC AAAATAAGCT GTTTGCCACC TC -            #AAAACTTT   1544                                                                 - - ATGAACTTCA CCACCACTAG TGTCTGTCCA TGGAGTTAGA GGGGACATCA CT -            #TAGAAGTT   1604                                                                 - - CTTATAGAAA GGACACAAGT TTGTTTCCTG GCTTTACCTT GGGAAAATGC TA -            #GCAACATT   1664                                                                 - - ATAGAAATTT TGCCTTGTTG CCTTATCTTC TTCCAAATGT ACTGTTAAAT AA -            #AAATAAAG   1724                                                                 - - GGTTACCCCA TGCAATCAAA AAAAAAAAAA AAAAA       - #                       - #     1759                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 251 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Lys Asn Phe Phe Leu Thr Asn Arg Ala Arg Gl - #u Arg Ser Asp Thr        Phe                                                                               1               5 - #                 10 - #                 15             - - Ile Asn Leu Arg Glu Val Leu Asn Arg Phe Ly - #s Leu Pro Pro Gly Glu                   20     - #             25     - #             30                  - - Tyr Ile Leu Val Pro Ser Thr Phe Glu Pro As - #n Lys Asp Gly Asp Phe               35         - #         40         - #         45                      - - Cys Ile Arg Val Phe Ser Glu Lys Lys Ala As - #p Tyr Gln Ala Val Asp           50             - #     55             - #     60                          - - Asp Glu Ile Glu Ala Asn Leu Glu Glu Phe As - #p Ile Ser Glu Asp Asp       65                 - # 70                 - # 75                 - # 80       - - Ile Asp Asp Gly Phe Arg Arg Leu Phe Ala Gl - #n Leu Ala Gly Glu Asp                       85 - #                 90 - #                 95              - - Ala Glu Ile Ser Ala Phe Glu Leu Gln Thr Il - #e Leu Arg Arg Val Leu                  100      - #           105      - #           110                  - - Ala Lys Arg Gln Asp Ile Lys Ser Asp Gly Ph - #e Ser Ile Glu Thr Cys              115          - #       120          - #       125                      - - Lys Ile Met Val Asp Met Leu Asp Ser Asp Gl - #y Ser Gly Lys Leu Gly          130              - #   135              - #   140                          - - Leu Lys Glu Phe Tyr Ile Leu Trp Thr Lys Il - #e Gln Lys Tyr Gln Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Tyr Arg Glu Ile Asp Val Asp Arg Ser Gl - #y Thr Met Asn Ser        Tyr                                                                                             165  - #               170  - #               175             - - Glu Met Arg Lys Ala Leu Glu Glu Ala Gly Ph - #e Lys Met Pro Cys Gln                  180      - #           185      - #           190                  - - Leu His Gln Val Ile Val Ala Arg Phe Ala As - #p Asp Gln Leu Ile Ile              195          - #       200          - #       205                      - - Asp Phe Asp Asn Phe Val Arg Cys Leu Val Ar - #g Leu Glu Thr Leu Phe          210              - #   215              - #   220                          - - Lys Ile Phe Lys Gln Leu Asp Pro Glu Asn Th - #r Gly Thr Ile Glu Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Leu Ile Ser Trp Leu Cys Phe Ser Val Le - #u                                          245  - #               250                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 700 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Met Ala Gly Ile Ala Ala Lys Leu Ala Lys As - #p Arg Glu Ala Ala        Glu                                                                             1               5   - #                10  - #                15              - - Gly Leu Gly Ser His Glu Arg Ala Ile Lys Ty - #r Leu Asn Gln Asp Tyr                  20      - #            25      - #            30                   - - Glu Ala Leu Arg Asn Glu Cys Leu Glu Ala Gl - #y Thr Leu Phe Gln Asp              35          - #        40          - #        45                       - - Pro Ser Phe Pro Ala Ile Pro Ser Ala Leu Gl - #y Phe Lys Glu Leu Gly          50              - #    55              - #    60                           - - Pro Tyr Ser Ser Lys Thr Arg Gly Met Arg Tr - #p Lys Arg Pro Thr Glu      65                  - #70                  - #75                  - #80        - - Ile Cys Ala Asp Pro Gln Phe Ile Ile Gly Gl - #y Ala Thr Arg Thr Asp                      85  - #                90  - #                95               - - Ile Cys Gln Gly Ala Leu Gly Asp Cys Trp Le - #u Leu Ala Ala Ile Ala                  100      - #           105      - #           110                  - - Ser Leu Thr Leu Asn Glu Glu Ile Leu Ala Ar - #g Val Val Pro Leu Asn              115          - #       120          - #       125                      - - Gln Ser Phe Gln Glu Asn Tyr Ala Gly Ile Ph - #e His Phe Gln Phe Trp          130              - #   135              - #   140                          - - Gln Tyr Gly Glu Trp Val Glu Val Val Val As - #p Asp Arg Leu Pro Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Asp Gly Glu Leu Leu Phe Val His Ser Al - #a Glu Gly Ser Glu        Phe                                                                                             165  - #               170  - #               175             - - Trp Ser Ala Leu Leu Glu Lys Ala Tyr Ala Ly - #s Ile Asn Gly Cys Tyr                  180      - #           185      - #           190                  - - Glu Ala Leu Ser Gly Gly Ala Thr Thr Glu Gl - #y Phe Glu Asp Phe Thr              195          - #       200          - #       205                      - - Gly Gly Ile Ala Glu Trp Tyr Glu Leu Lys Ly - #s Pro Pro Pro Asn Leu          210              - #   215              - #   220                          - - Phe Lys Ile Ile Gln Lys Ala Leu Gln Lys Gl - #y Ser Leu Leu Gly Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Ile Asp Ile Thr Ser Ala Ala Asp Ser Gl - #u Ala Ile Thr Phe        Gln                                                                                             245  - #               250  - #               255             - - Lys Leu Val Lys Gly His Ala Tyr Ser Val Th - #r Gly Ala Glu Glu Val                  260      - #           265      - #           270                  - - Glu Ser Asn Gly Ser Leu Gln Lys Leu Ile Ar - #g Ile Arg Asn Pro Trp              275          - #       280          - #       285                      - - Gly Glu Val Glu Trp Thr Gly Arg Trp Asn As - #p Asn Cys Pro Ser Trp          290              - #   295              - #   300                          - - Asn Thr Ile Asp Pro Glu Glu Arg Glu Arg Le - #u Thr Arg Arg His Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Gly Glu Phe Trp Met Ser Phe Ser Asp Ph - #e Leu Arg His Tyr        Ser                                                                                             325  - #               330  - #               335             - - Arg Leu Glu Ile Cys Asn Leu Thr Pro Asp Th - #r Leu Thr Ser Asp Thr                  340      - #           345      - #           350                  - - Tyr Lys Lys Trp Lys Leu Thr Lys Met Asp Gl - #y Asn Trp Arg Arg Gly              355          - #       360          - #       365                      - - Ser Thr Ala Gly Gly Cys Arg Asn Tyr Pro As - #n Thr Phe Trp Met Asn          370              - #   375              - #   380                          - - Pro Gln Tyr Leu Ile Lys Leu Glu Glu Glu As - #p Glu Asp Glu Glu Asp      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gly Glu Ser Gly Cys Thr Phe Leu Val Gly Le - #u Ile Gln Lys His        Arg                                                                                             405  - #               410  - #               415             - - Arg Arg Gln Arg Lys Met Gly Glu Asp Met Hi - #s Thr Ile Gly Phe Gly                  420      - #           425      - #           430                  - - Ile Tyr Glu Val Pro Glu Glu Leu Ser Gly Gl - #n Thr Asn Ile His Leu              435          - #       440          - #       445                      - - Ser Lys Asn Phe Phe Leu Thr Asn Arg Ala Ar - #g Glu Arg Ser Asp Thr          450              - #   455              - #   460                          - - Phe Ile Asn Leu Arg Glu Val Leu Asn Arg Ph - #e Lys Leu Pro Pro Gly      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Glu Tyr Ile Leu Val Pro Ser Thr Phe Glu Pr - #o Asn Lys Asp Gly        Asp                                                                                             485  - #               490  - #               495             - - Phe Cys Ile Arg Val Phe Ser Glu Lys Lys Al - #a Asp Tyr Gln Ala Val                  500      - #           505      - #           510                  - - Asp Asp Glu Ile Glu Ala Asn Leu Glu Glu Ph - #e Asp Ile Ser Glu Asp              515          - #       520          - #       525                      - - Asp Ile Asp Asp Gly Val Arg Arg Leu Phe Al - #a Gln Leu Ala Gly Glu          530              - #   535              - #   540                          - - Asp Ala Glu Ile Ser Ala Phe Glu Leu Gln Th - #r Ile Leu Arg Arg Val      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Leu Ala Lys Arg Gln Asp Ile Lys Ser Asp Gl - #y Phe Ser Ile Glu        Thr                                                                                             565  - #               570  - #               575             - - Cys Lys Ile Met Val Asp Met Leu Asp Ser As - #p Gly Ser Gly Lys Leu                  580      - #           585      - #           590                  - - Gly Leu Lys Glu Phe Tyr Ile Leu Trp Thr Ly - #s Ile Gln Lys Tyr Gln              595          - #       600          - #       605                      - - Lys Ile Tyr Arg Glu Ile Asp Val Asp Arg Se - #r Gly Thr Met Asn Ser          610              - #   615              - #   620                          - - Tyr Glu Met Arg Lys Ala Leu Glu Glu Ala Gl - #y Phe Lys Met Pro Cys      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Gln Leu His Gln Val Ile Val Ala Arg Phe Al - #a Asp Asp Gln Leu        Ile                                                                                             645  - #               650  - #               655             - - Ile Asp Phe Asp Asn Phe Val Arg Cys Leu Va - #l Arg Leu Glu Thr Leu                  660      - #           665      - #           670                  - - Phe Lys Ile Phe Lys Gln Leu Asp Pro Glu As - #n Thr Gly Thr Ile Glu              675          - #       680          - #       685                      - - Leu Asp Leu Ile Ser Trp Leu Cys Phe Ser Va - #l Leu                          690              - #   695              - #   700                        __________________________________________________________________________

What is claimed is:
 1. An isolated protein having IL-1-R intracellularligand protein activity and comprising the amino acid sequence of SEQ IDNO:2 said protein being substantially free from other mammalianproteins.
 2. A composition comprising the protein of claim 1 and apharmaceutically acceptable carrier.
 3. An isolated protein havingIL-1-R intracellular ligand protein activity and comprising the aminoacid sequence of SEQ ID NO:4 said protein being substantially free fromother mammalian proteins.
 4. A composition comprising the protein ofclaim 3 and a pharmaceutically acceptable carrier.
 5. An IL-1-Rintracellular ligand protein produced according to a methodcomprising:(1) growing a culture of a host cell transformed with apolynucleotide which is operably linked to an expression controlsequence in a suitable culture medium; and (2) purifying said IL-1-Rintracellular ligand protein from the culture wherein saidpolynucleotide encodes a protein having IL-1-R intracellular ligandprotein activiy, and wherein said polynucleotide is selected from thegroup consisting of:(a) a polynucleotide comprising the nucleotidesequence of SEQ ID NO:1 from nucleotide 2 to nucleotide 529; and (b) apolynucleotide encoding an IL-1-R intracellular ligand proteincomprising the amino acid sequence of SEQ ID NO:2.
 6. A compositioncomprising the protein of claim 5 and a pharmaceutically acceptablecarrier.
 7. The protein of claim 5, wherein said polynucleotidecomprises the nucleotide sequence of SEQ ID NO:1 from nucleotide 2 tonucleotide
 529. 8. The protein of claim 5, wherein said polynucleotidecomprises a polynucleotide encoding a protein comprising the amino acidsequence of SEQ ID NO:2.
 9. An IL-1-R intracellular ligand proteinproduced according to a method comprising:(1) growing a culture of ahost cell transformed with a polynucleotide which is operably linked toan expression control sequence in a suitable culture medium; and (2)purifying said IL-1-R intracellular ligand protein from the culturewherein said polynucleotide encodes a protein having IL-1-Rintracellular ligand protein activity, and wherein said polynucleotideis selected from the group consisting of:(a) a polynucleotide comprisingthe nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide961; and (b) a polynucleotide encoding an L-1-R intracellular ligandprotein comprising the amino acid sequence of SEQ ID NO:4.
 10. Acomposition comprising the protein of claim 9 and a pharmaceuticallyacceptable carrier.
 11. The protein of claim 9, wherein saidpolynucleotide comprises the nucleotide sequence of SEQ ID NO:3 fromnucleotide 2 to nucleotide
 961. 12. The protein of claim 9, wherein saidpolynucleotide comprises a polynucleotide encoding a protein comprisingthe amino acid sequence of SEQ ID NO:4.